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目的克隆弓形虫速殖子特异表达蛋白SAG1的启动子序列,为弓形虫基因操作提供工具。方法应用PCR方法扩增弓形虫速殖子特异表达蛋白SAG1的5′非编码区、致密颗粒蛋白GRA2的3′编码区和红色荧光蛋白(RFP)基因,并构建重组质粒pMD/SAG-RFP-GRA,经电穿孔法将其转染弓形虫速殖子,倒置荧光显微镜观察RFP的表达。结果重组质粒pMD/SAG-RFP-GRA经双酶切和测序证明构建正确,质粒转染弓形虫速殖子24h,荧光显微镜下可观察到红色荧光,表明克隆的SAG1启动子具有转录活性。结论已成功克隆了弓形虫速殖子特异表达蛋白SAG1的启动子序列,并能介导外源基因在弓形虫体内的表达。
Objective To clone the promoter sequence of tachyzoites specific expression protein SAG1 and provide a tool for the genetic manipulation of Toxoplasma gondii. Methods The 5 ’noncoding region of tachyzoites specific gene SAG1, the 3’ coding region of GRA2 and the red fluorescent protein (RFP) gene were amplified by PCR. The recombinant plasmid pMD / SAG-RFP- GRA. The Toxoplasma gondii tachyzoites were transfected by electroporation, and the expression of RFP was observed by inverted fluorescence microscope. Results The recombinant plasmid pMD / SAG-RFP-GRA was confirmed by double enzyme digestion and sequencing. The plasmid was transfected with tachyzoites of Toxoplasma gondii for 24 hours, and the red fluorescence was observed under fluorescence microscope. The results showed that the cloned SAG1 promoter had transcriptional activity. Conclusion The promoter sequence of tachyzoites specific expression gene SAG1 was successfully cloned and the expression of foreign gene in Toxoplasma gondii was also mediated.